1. Field of the Invention
The present invention relates generally to protein chemistry. More specifically, the present invention relates to the identification of inhibitors of the heterodimeric cytotoxin ricin, using computer modeling of the ricin active site as a template in structure-based drug design. Moreover, the present invention relates to the use of the identified inhibitors as antidotes to ricin or to facilitate immunotoxin treatment by controlling non-specific cytotoxicity. In addition, Shiga toxin, a compound essentially identical to the protein originally isolated from Shigella dysenteriae, has an active protein chain that is a homologue of the ricin active chain, and catalyzes the same depurination reaction. Thus, the present invention is drawn additionally to identifying inhibitors as antidotes to Shiga toxin.
2. Description of the Related Art
Ricin is a potent he terodimeric cytotoxin easily isolated from the seeds of the castor plant, Ricinus communis. The protein consists of a lectin B chain, which can bind cell surfaces and is linked by disulfide bonds to an Achain (RTA), which enzymatically depurinates a key adenine residue in 28 S rRNA (Endo and Tsurugi, J. Biol. Chem., 262:8128-30 (1987)). The molecular structure of ricin is represented in FIGS. 1A and 1B while the adenine depurination reaction mechanism is presented in FIG. 2.
Ricin is an extraordinarily toxic molecule that attacks ribosomes, thereby inhibiting protein synthesis. Ricin has an LD50 of approximately 1 xcexcg/kg body weight for mice, rats, and dogs and is ten times more potent against rabbits (Olsnes, S. and Pihl, A., xe2x80x9cToxic Lectins and Related Proteins,xe2x80x9d The Molecular Action of Toxins and Viruses, pp 52-105 (1982)). The toxic dose for humans is likely to be in the xcexcg/kg range which ranks it among the most toxic substances known.
The protein has been used extensively in the design of therapeutic immunotoxins. In these constructs, ricin, RTA, or a related toxin is chemically or genetically linked to an antibody to form a so-called xe2x80x9cmagic bulletxe2x80x9d, which can target preferentially those cell lines carrying antigenic markers recognized by the antibody (Frankel, A.E., ed., Immunotoxins, Kluwer academic publishers, Boston (1988)).
Ricin also has been used as a poison agent. The protein gained notoriety when it was used in the famous xe2x80x9cumbrella tipxe2x80x9d assassination of Georgy Markov and was also used in an unsuccessful attempt to poison the Soviet dissident Alexander Solzhenitsyn. More recently, ricin was prepared by a militant anti-tax group which planned to poison IRS personnel.
Given the above, there is interest in identifying or designing potent inhibitors of ricin. These inhibitors could, in principle, be used to facilitate immunotoxin treatment by helping to control nonspecific cytotoxicity, or could be used as antidotes to poison attacks. Recently there has been interest in structure-based drug designxe2x80x94that is, using the knowledge of protein structure to identify enzyme inhibitors. The most common paradigm for this overall process has been called an xe2x80x9citerative protein crystallographic algorithmxe2x80x9d by Appelt et al., J. Med. Chem. 34:1925-34 (1991), where the design of inhibitors for thymidylate synthetase is described. The main concept of the approach is that the protein active site is used as a template to design or to identify complementary ligands. The identified putative ligands are ranked and tested kinetically. Promising inhibitor candidates are bound to the protein target and analyzed crystallographically for comparison with the proposed model. Additionally, alterations are made in the inhibitor to improve binding and a new round of tests is carried out on the altered compound.
A number of laboratories have used variations on this protocol to design efficacious inhibitors. For example, the search program DOCK (see Kuntz et al., J. Mol. Biol. 161:269-88 (1982)) was used to predict that the known anti-psychotic drug haloperidol would bind to the HIV protease (DesJarlais et al., Proc. Natl. Acad. Sci USA 87:6644-48 (1990)). Crystallographic studies together with computer aided search methods also were used in the design of inhibitors of purine nucleoside phosphorylase. The program GRID (Goodford, P. J., J. Med. Chem. 28:849-57 (1985)) has been used to design two very successful inhibitors of influenza virus. Certain chemical substitutions to the sialic acid substrate were predicted to be energetically favorable, based on interactions with the known X-ray structure of the enzyme. Subsequent binding assays revealed Ki values as low as 0.2 nM. These compounds not only inhibited neuraminidase but retarded viral replication in cultured cell and animal models as well.
The X-ray structure of ricin has been solved (Montfort et al., J. Biol. Chem. 262:5398-03 (1987)), refined to 2.5 xc3x85 (Rutenber et al., Proteins 10:240-50 (1991)), and described in detail (Katzin et al., Proteins 10:251-59 (1991); and Rutenber and Robertus, Proteins 10:260-69 (1991)). The structure of RTA pressed in Escherichia coli has been resolved to 2.3 xc3x85 resolution for monoclinic crystals (Mlsna et al., Prot. Sci. 2:429-35 (1993)), and recently to 1.8 xc3x85 resolution for a tetragonal form (Weston et al., J. Mol. Biol. 244:410-422 (1994)).
The X-ray model of RTA allowed identification of a number of amino acids which were hypothesized to be important for substrate binding and for the depurination mechanism; these residues include Glu 177, Arg 180, Trp 211, Tyr 80 and Tyr 123. Site-directed mutagenesis of the cloned RTA gene has been used to examine the relative significance of these residues (see, e.g., Schlossman et al., Mol. Cell. Biol. 9:5012-21 (1989); Frankel et al., Mol. Cell. Biol. 10:6257-63 (1990); Ready et al., Proteins 10:270-78 (1991); and Kim and Robertus, Protein Engineering 5:775-79 (1992)). In addition, Monzingo and Robertus, J. Mol. Biol. 227:1136-45 (1992), carried out an X-ray analysis of substrate analogs in the RTA active site, examining FMP, adenyl guanosine (ApG) and guanyl adenosine (GpA). The structure of important substrate and analog bases, together with the numbering scheme used in energy minimizations, is shown in FIGS. 8A-8D.
Several closely related mechanisms of action have been proposed which incorporate elements of both the structural and kinetic analyses. It is likely that the susceptible adenine base binds between tyrosines 80 and 123 while forming specific hydrogen bonds with the backbone carbonyl and amido nitrogen of Val 81 and with the carbonyl of Gly 121. In the hydrolysis, the leaving adenine is at least partially protonated by Arg 180, and Glu 177 may stabilize a putative oxycarbonium transition state or, more likely, act as a base to polarize the attacking water.
Shiga toxin, a compound essentially identical to the protein originally isolated from Shigella dysenteriae, has an A chain which is activated by proteolysis, generating an active A1 enzyme and an A2 fragment, which remains bound until a disulfide bond linking them is reduced. The active A1 chain (STA1) is a homologue of RTA, and catalyzes the same depurination reaction. E. coli strains can carry the gene for Shiga toxin, and this renders them pathogenic. Human infection by Shiga toxin lead to hemorrhagic colitis and hemolytic-uremic syndromexe2x80x94commonly referred to as xe2x80x9chamburger diseasexe2x80x9dxe2x80x94a severe and often fatal form of food poisoning. It is difficult to control outbreaks of hamburger disease because antibiotics tend to lyse the bacteria, releasing the destructive toxin into the system, aggravating tissue damage and internal bleeding. An effective inhibitor of STA1 would be a powerful adjunct to treatment of hemorrhagic colitis and hemolytic-uremic syndrome.
The Shiga toxin gene has been cloned and engineered to express the enzyme. A comparison of the amino acid sequences of RTA and STA show clearly that they are homologues. An energy-minimized model of STA1 was constructed (Deresiewicz et al, Biochemistry 31:3272-80 (1992)), and it was noted that key residues conserved among plant Ribosome Inactivating Protein (RIP) enzymes are conserved in STA1 (Katzin et al., Proteins 10:251-59 (1991)).
Until the present invention, no inhibitors for RTA or STA1 had been identified. Even FMP, which is known to bind RTA, is not an effective inhibitor of the RTA. Thus, the prior art is deficient in identifying compounds which are effective inhibitors of ricin. The present invention fulfills this long-standing need and desire in the art.
The present invention provides compounds that are effective inhibitors of the cytotoxic proteins ricin or Shiga toxin.
In one object of the present invention, there is provided a compound effective for inhibiting ricin, the compound able to act within an active site of RTA and having an aromatic heterocyclic molecular core, wherein the aromatic heterocyclic molecular core resembles an adenine moiety in size and shape and is derivatized with at least one polar substituent such that the polar substituent interacts in the active site of RTA. In another embodiment of this object of the invention there is provided a compound wherein said polar substituent is an amine group able to donate hydrogen bonds to a carbonyl oxygen of Val 81 or Gly 121. As another embodiment of this object of the present invention, there is provided a compound wherein the inhibitor further comprises at least one pendant group which binds an amino acid adjacent to the active site. In addition, the inhibitor further may comprise at least one moiety which reacts with a shallow channel in the RTA chain.
In another object of the present invention, there is provided a compound effective for inhibiting ricin, the compound being able to act within an active site of RTA, having nonpolar interactions with a side chain of an amino acid in the active site selected from the group Tyr 80, Ile 172 or Tyr 123, and having polar interactions with a side chain of an amino acid in the active site, selected from the group of carbonyl oxygens; of Gly 121 or Val 81, backbone amides of Val 81 or Tyr 123, or atoms on side chains of Arg 180, Tyr 80, Tyr 123 or Asn 78. In addition, an embodiment of the present object provides an inhibitor further comprising at least one nonpolar moiety which interacts with side chains from Trp 211, Leu 45, Val 256, Tyr 257 or Thr 77 of said RTA chain. Alternatively, the inhibitor may possess at least one polar moiety which interacts with the carbonyl oxygens of Thr 77 or Tyr 257, or side chains from Asn 47 and Arg 258 of the RTA chain, or further comprise at least one polar moiety which interacts with an amino acid from a second pocket of said RTA chain. In addition, the inhibitor may interact in a nonpolar fashion with side chains from amino acids selected from the group of Tyr 80, Val 82, Phe 57, Thr 77 and Arg 56.
In yet another object of the present invention, there is provided a compound effective for inhibiting ricin, wherein said compound is a pteroic acid analog.
In an additional object of the present invention, there is provided a compound effective for inhibiting Shiga toxin, the compound able to act within an active site of STA1 and having an aromatic heterocyclic molecular core, wherein the aromatic heterocyclic molecular core resembles an adenine moiety in size and shape and is derivatized with polar substituents such that the polar substituents interact in the active site of RTA.
An additional object of the present invention, there is provided a compound effective for inhibiting Shiga toxin, wherein said compound is a pteroic acid analog.
Additionally, the present invention provides a method for identifying a compounds effective for inhibiting ricin, comprising the steps of performing at least one technique from the group of molecular modeling, crystallography, and energy minimization; protein synthesis assay; and phage display, and a method for identifying a compounds effective for inhibiting Shiga toxin, comprising the steps of performing at least one technique from the group of molecular modeling, crystallography, and energy minimization; protein synthesis assay; and phage display.
Another object of the present invention is to provide the pteroic acid analog as a pharmaceutical compound as an antidote to ricin or to facilitate immunotoxin treatment by controlling non-specific cytotoxicity.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.